Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography

Background Overexpressed in various solid tumors, gastrin-releasing peptide receptor (GRPR) is a promising cancer imaging marker and therapeutic target. Although antagonists are preferable for the development of GRPR-targeted radiopharmaceuticals due to potentially fewer side effects, internalization of agonists may lead to longer tumor retention and better treatment efficacy. In this study, we systematically investigated unnatural amino acid substitutions to improve in vivo stability and tumor uptake of a previously reported GRPR-targeted agonist tracer, [68Ga]Ga-TacBOMB2 (68Ga-DOTA-Pip-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Thz14-NH2). Results Unnatural amino acid substitutions were conducted for Gln7, Trp8, Ala9, Val10, Gly11 and His12, either alone or in combination. Out of 25 unnatural amino acid substitutions, tert-Leu10 (Tle10) and NMe-His12 substitutions were identified to be preferable modifications especially in combination. Compared with the previously reported [68Ga]Ga-TacBOMB2, the Tle10 and NMe-His12 derived [68Ga]Ga-LW01110 showed retained agonist characteristics and improved GRPR binding affinity (Ki = 7.62 vs 1.39 nM), in vivo stability (12.7 vs 89.0% intact tracer in mouse plasma at 15 min post-injection) and tumor uptake (5.95 vs 16.6 %ID/g at 1 h post-injection). Conclusions Unnatural amino acid substitution is an effective strategy to improve in vivo stability and tumor uptake of peptide-based radiopharmaceuticals. With excellent tumor uptake and tumor-to-background contrast, [68Ga]Ga-LW01110 is promising for detecting GRPR-expressing cancer lesions with PET. Since agonists can lead to internalization upon binding to receptors and foreseeable long tumor retention, our optimized GRPR-targeted sequence, [Tle10,NMe-His12,Thz14]Bombesin(7–14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents. Supplementary Information The online version contains supplementary material available at 10.1186/s41181-024-00241-7.


Background
Gastrin-releasing peptide receptor (GRPR) is a G protein-coupled receptor, expressed in pancreas, gastrointestinal tract, and central nervous system, and involved in physiological functions such as synaptic plasticity, hormone secretion, and smooth muscle contraction (Jensen et al. 2008;Bitar and Zhu 1993;Weber 2009).Overexpression of GRPR has been reported to induce cancer cell proliferation and facilitate malignant neoplasm development (Jensen et al. 2008;Weber 2009;Cornelio et al. 2007;Hajri et al. 1996;Moody et al. 1996;Preston et al. 1995Preston et al. , 1994;;Gugger and Reubi 1999;Markwalder and Reubi 1999;Roesler et al. 2006;Shimoda 1992;Qin et al. 1994).The overexpression of GRPR in various tumors makes it a promising target for the design of targeted radiopharmaceuticals for diagnosis and radioligand therapy of GRPR-expressing cancers.
The development of GRPR-targeted radiopharmaceuticals has been focused on using antagonist sequences as targeting vectors because of their potentially higher tumor uptake due to higher in vivo stability (Ghosh et al. 2019) and more binding sites than those available for agonists (Mansi et al. 2009), and/or less short term adverse effects (Chatalic et al. 2016;Mansi et al. 2013).However, agonists can be internalized upon binding to GRPR and lead to a longer tumor retention (Jensen et al. 2008;Mansi et al. 2013;Yang et al. 2011), which might be preferable especially for the development of radiotherapeutic agents.The in vivo instability of GRPR-targeted ligands is caused by enzymatic degradation especially by neutral endopeptidase 24.11 (NEP) (Chatalic et al. 2016;Nock et al. 2014).The reported cleavage sites including His 12 -Leu 13 , Trp 8 -Ala 9 and Gln 7 -Trp 8 for AMBA derivatives and Trp 8 -Ala 9 , Ala 9 -Val 10 and Gln 7 -Trp 8 for RM2 derivatives (Kähkönen et al. 2013;Linder et al. 2009).
We hypothesized that (1) replacing amino acids at the potential cleavage sites of our previously reported GRPR agonist [ 68 Ga]Ga-TacBOMB2 ([ 68 Ga]Ga-DOTA-Pip-D-Phe 6 -Gln 7 -Trp 8 -Ala 9 -Val 10 -Gly 11 -His 12 -Leu 13 -Thz 14 -NH 2 ) with unnatural amino acids can improve in vivo stability and retain the agonist characteristics; and (2) the resulting stabilized [ 68 Ga]Ga-TacBOMB2 derivatives can also retain the minimal pancreas uptake characteristics.Thus, in this study we first synthesized the GRPR-targeted sequence of TacBOMB2 (LW01085, D-Phe 6 -Gln 7 -Trp 8 -Ala 9 -Val 10 -Gly 11 -His 12 -Leu 13 -Thz 14 -NH 2 , Fig. 1) and systematically substituted the amino acids (Gln 7 , Trp 8 , Ala 9 , Val 10 , Gly 11 and His 12 ) at its potential cleavage sites with an unnatural amino acid.The derivatives with high GRPR binding affinity were coupled with the DOTA chelator and 4-amino-(1-carboxymethyl)piperidine (Pip) linker.The binding affinities of their Ga-complexed standards were further confirmed by in vitro competition assays, and their agonists characteristics were confirmed by calcium release assays.Finally, the lead candidates were radiolabeled with 68 Ga and evaluated by PET imaging and ex vivo biodistribution studies using the GRPR-expressing PC-3 prostate cancer model.

Synthesis of GRPR-targeted ligands
Detailed procedures for the synthesis, purification, and characterization of GRPRtargeted ligands and their nat Ga/ 68 Ga-complexed analogs are provided in the Supplementary Information (Additional file 1: Figs.S1-S46 and Tables S1-S4).

In vitro competition binding assays
Inhibition constants (K i ) of GRPR-targeted ligands to GRPR were measured by in vitro competition binding assay using PC-3 cells and [ 125 I-Tyr 4 ]Bombesin as the radioligand following previously published procedures (Wang et al. 2022(Wang et al. , 2023;;Bratanovic et al. 2022).The assays were conducted in triplicate with varied concentrations (10 μM to 1 pM) of tested ligands.Briefly, PC-3 cells were seeded in 24-well poly-D-lysine plates at 2 × 10 5 cells/well 24-48 h prior to the assay.The growth medium was replaced with 400 μL of reaction medium (RPMI 1640 containing 2 mg/ mL BSA, and 20 mM HEPES), then the plates were incubated at 37 °C for 60 min.The tested ligands in 50 μL reaction medium and 50 μL of 0.01 nM [ 125 I-Tyr 4 ]Bombesin were added into the wells followed by incubation with moderate agitation for 1 h at 37 °C.Cells were gently washed with ice-cold DPBS twice, harvested by trypsinization, and counted for radioactivity on a Perkin Elmer (Waltham, MA, USA) Wizard2 2480 automatic gamma counter.Data were analyzed using nonlinear regression with GraphPad (San Diego, CA, USA) Prism 8 software.

Fluorometric calcium release assays
Following previously published procedures (Bratanovic et al. 2022;Lau et al. 2019), 5 × 10 4 PC-3 cells were seeded in 96-well clear bottom black plates 24 h prior to the assay.The growth medium was removed and replaced with a loading buffer containing a calcium-sensitive dye (FLIPR Calcium 6 assay kit from Molecular Devices, San Jose, CA, USA).After incubated at 37 °C for 30 min, the plates were placed in a Flex-Station 3 microplate reader (Molecular Devices).Tested ligands (50 nM) or DPBS (negative control) were added to the cells and the fluorescent signals were acquired for 2 min.Agonistic/antagonistic properties of the tested ligands were determined based on the relative fluorescent unit (RFU = max -min) of their generated fluorescent signals.

LogD 7.4 measurements
The LogD 7.4 values of 68 Ga-labeled tracers were measured using the shake flask method as previously published (Lin et al. 2015).Briefly, aliquots (2 μL) of the 68 Ga-labeled tracers were added into a 15 mL falcon tube containing 3 mL of n-octanol and 3 mL of DPBS (pH 7.4).The mixture was vortexed for 1 min and then centrifuged at 3,000 rpm for 15 min.Samples of the n-octanol (1 mL) and DPBS (1 mL) layers were collected and measured in a Perkin Elmer Wizard2 2480 automatic gamma counter.LogD 7.4 was calculated with the following equation: LogD 7.4 = log 10 [(counts in n-octanol phase)/(counts in DPBS phase)].

Biodistribution, PET imaging, and in vivo stability studies
PET/CT imaging, biodistribution, and in vivo stability studies were conducted on male NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl /SzJ (NRG) mice following previously published procedures (Bratanovic et al. 2022;Lau et al. 2019;Lin et al. 2015;Kuo et al. 2018).The experiments were conducted according to the guidelines established by the Canadian Council on Animal Care and approved by Animal Ethics Committee of the University of British Columbia.The mice were anaesthetized by inhalation of 2.5% isoflurane in 2 mL/ min oxygen, and implanted subcutaneously with 5 × 10 6 PC-3 cells (100 µL; 1:1 PBS/ Matrigel) behind the left shoulder.Mice were used for PET/CT imaging and biodistribution studies when the tumor grew to 5-8 mm in diameter over around 4 weeks.
PET imaging experiments were conducted using a Siemens Inveon (Knoxville, TN, USA) micro PET/CT scanner.Each tumor bearing mouse was injected with 3-5 MBq (90.6-166.8ng) of 68 Ga-labeled tracer via the lateral caudal tail vein under anaesthesia (2% isoflurane in oxygen).For blocking, the mice were co-injected with 100 μg of [D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 ]Bombesin(6-14).The mice were allowed to recover and roam freely in their cages.After 50 min, the mice were sedated again with 2% isoflurane in oxygen inhalation and positioned on the scanner.A 10-min CT scan was conducted first for localization and attenuation correction after segmentation for reconstructing the PET images, followed by a 10-min static PET imaging acquisition.
For biodistribution studies, the mice were injected with 2-4 MBq (50.2-106.8ng) of radiotracer as described above.For blocking, the mice were co-injected with [D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 ]Bombesin(6-14) (100 μg).At 1 h post-injection, the mice were anesthetized with 2% isoflurane inhalation, and euthanized by CO 2 inhalation.Blood was withdrawn by cardiac puncture, and organs/tissues of interest were collected.The collected organs/tissues were rinsed with PBS, blotted dry, weighed, and counted using the automatic gamma counter.
For in vivo stability studies, the 68 Ga-labeled ligand (6-15 MBq) was injected via the lateral caudal vein into healthy male NRG mice (n = 3).At 15 min post-injection, mice were euthanized, and the urine and blood samples were collected.The plasma was extracted from whole blood samples by the addition of CH 3 CN (500 μL), 1-min vortex, 20-min centrifugation, and the separation of supernatant.The plasma and urine samples were analyzed via radio-HPLC using the conditions for quality control (Additional file 1: Table S4).

Statistical analysis
Statistical analyses were performed by Student's t-test using the Microsoft (Redmond, WA, USA) Excel software.The comparison of biodistribution data between two tracers was conducted via unpaired two-tailed test.Unpaired one-tailed test was used to compare biodistribution data between blocked/unblocked mice injected with the same tracer.The difference was considered statistically significant when the p value was < 0.05.

PET imaging and biodistribution
The capability of 68 Ga-labeled TacBOMB2 derivatives to target GRPR in vivo was evaluated by PET imaging and biodistribution studies in mice bearing GRPR-expressing PC-3 tumor xenografts.As shown in Fig. 4, all 68 Ga-labeled tracers enabled visualization of PC-3 tumors with good tumor-to-background contrasts.These tracers were excreted mainly via the renal pathway and had only low to moderate uptake in pancreas.Higher tumor uptake was observed by using [ 68 Ga]Ga-LW01110, [ 68 Ga]Ga-LW02040 and [ 68 Ga] Ga-LW01142, followed by [ 68 Ga]Ga-LW01107 and [ 68 Ga]Ga-LW01108, and [ 68 Ga] Ga-LW02021 had the lowest tumor uptake.[ 68 Ga]Ga-LW01142 which showed high blood retention at 1 h post-injection was further evaluated at 3 h post-injection (Fig. 4F).The tumor uptake of [ 68 Ga]-LW01142 increased further at 3 h post-injection, leading to an enhanced tumor-to-background contrast.Co-injection of [D-Phe 6 ,Leu-NHEt 13 ,des-Met 14 ]Bombesin(6-14) reduced tumor uptake of both [ 68 Ga]Ga-LW01110 and [ 68 Ga] Ga-LW01142 at 1 h post-injection (Figs.4C and F).
Despite being popularly used for the design of GRPR-targeted antagonist ligands (Richter et al. 2016;Sah et al. 2015), NMe-Gly 11 substitution was reported to cause > 30-fold reduction in GRPR binding affinity for an agonist sequence (K i = 0.7 vs 25 nM for Ac-Bombesin(7-14) and Ac-[NMe-Gly 11 ]Bombesin(7-14), respectively) (Horwell et al. 1996).Consistent with the previous report, we also observed a dramatic reduction in GRPR binding affinity with the NMe-Gly 11 substitution (Figs.2F and G, K i = 3.19 vs 12,790 nM for Ga-LW01142 and Ga-LW01143, respectively).αMe-Trp 8 substitution has been successfully used by the Wester group for the design of potent and stable radiolabeled GRPR-targeted antagonists derived from RM2 (Guenther et al. 2022).However, for agonist Ga-TacBOMB2, αMe-Trp 8 substitution in Ga-LW01149 caused significant loss of binding affinity (K i = 7.62 vs 342 nM, Figs.2A and D).Our data suggest that GRPR agonists and antagonists might bind to the receptors in different configurations as Data are presented as mean ± SD (n = 3).The data of [ 68 Ga]Ga-TacBOMB2, [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-AMBA have been reported previously (Wang et al. 2022(Wang et al. , 2023) )  αMe-Trp 8 and NMe-Gly 11 substitutions which are commonly used for antagonist modifications hinder the binding of agonists to the receptors.His 7 (the amino acid at the corresponding position in GRP), 2-Me-Trp 8 , 7-F-Trp 8 , 5-Me-Trp 8 , Tle 10 and NMe-His 12 substitutions, either alone or in combination, still led to GRPR-targeted ligands with potent binding affinities (K i = 1.34-14.9nM, Fig. 2).This suggests that compared with the targeted peptide sequences presented in Fig. 1, the addition of Ga-DOTA complex and the Pip linker does not affect their binding affinity.Similarly, based on the results of calcium release assays (Fig. 3), His 7 , 2-Me-Trp 8 , 7-F-Trp 8 , 5-Me-Trp 8 , Tle 10 and NMe-His 12 substitutions, either alone or in combination, do not change their agonist characteristics.
Subsequently, we radiolabeled potent candidates and evaluated their potential for prostate cancer imaging.As shown in Fig. 4, all 68 Ga-labeled tracers were successfully used to visualize PC-3 tumor xenografts in their PET images, confirming good GRPR targeting capabilities of these tracers.A lower tumor uptake was observed for [ 68 Ga] Ga-LW02021, which could be due to its relatively weaker GRPR binding affinity compared with those of others (K i = 13.6 vs 1.34 -3.19 nM, Fig. 2).The clearance of these tracers was mainly via the renal pathway, consistent with the highly hydrophilic nature of these tracers (LogD 7.4 values ≤ -1.81).A higher blood retention was observed for [ 68 Ga] Ga-LW01142 at 1 h post-injection, which could be due to its relatively higher lipophilicity than other tracers (LogD 7.4 = -1.81vs -2.46 --3.10,Table 1).
In vivo stability studies were subsequently conducted to verify our hypothesis.As shown in Table 1, compared with the previously reported [ 68 Ga]Ga-TacBOMB2 (12.7 ± 2.93% intact tracer at 15 min post-injection), Tle 10 and NMe-His 12 substitutions led to [ 68 Ga]Ga-LW01108 (35.3 ± 0.93% intact) and [ 68 Ga]Ga-LW01107 (66.2 ± 12.4% intact) with an improved in vivo stability.Combination of at least both Tle 10 and NMe-His 12 substitutions further led to [ 68 Ga]Ga-LW01110, [ 68 Ga]Ga-LW01142 and [ 68 Ga] Ga-LW02040 with an average ≥ 89% intact tracer at 15 min post-injection.These data are consistent with the trend of their tumor uptake observed from the ex vivo biodistribution studies: [ 68 Ga]Ga-TacBOMB2 ≈ [ 68 Ga]Ga-LW01108 < [ 68 Ga]Ga-LW01107 < [ 68 Ga] Ga-LW01110, [ 68 Ga]Ga-LW01142 and [ 68 Ga]Ga-LW02040.In addition, our in vivo stability data also suggest that His 12 -Leu 13 is the major cleavage site of GRPR-targeted ligands, followed by Ala 9 -Val 10 , and then Gln 7 -Trp 8 /Trp 8 -Ala 9 .This is also consistent with the fact that most of reported GRPR-targeted radioligands had modifications to avoid the cleavage at His 12 -Leu 13 such as using Sta 13 substitution for the RM2 derivatives (Kähkönen et al. 2013;Mansi et al. 2011) and Leu 13 ψThz 14 in our previously reported TacsBOMB derivatives (Wang et al. 2022).Contrary to the improved stability observed in plasma, no intact tracer was detected in urine samples even for ligands with both Tle 10 and NMe-His 12 substitutions (Additional file 1: Figs.S49-S53).This is due to the facts that GRPR-targeted ligands are cleaved mainly by NEP and kidneys have the highest NEP expression level (Jiang et al. 2004).Therefore, GRPR-targeted tracers which remain intact in plasma are completely metabolized by NEP in kidneys before being excreted into the urinary bladder.
Compared with the clinically validated [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-AMBA (Wang et al. 2022(Wang et al. , 2023)), our stabilized tracers ([ 68 Ga]Ga-LW01110, [ 68 Ga]Ga-LW01142 and [ 68 Ga]Ga-LW02040) have not only higher tumor uptake, but also comparable or even higher tumor-to-background uptake ratios (Additional file 1: Tables S2-S3).Most importantly, they also have a much lower pancreas uptake than [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-AMBA (4.40-11.7 vs 41.9-62.4%ID/gat 1 h post-injection).Therefore, these tracers are expected to have a higher sensitivity for detecting cancer lesions in or adjacent to the pancreas, and can achieve better treatment efficacy and cause less damage to the pancreas when radiolabeled with an α-or β-emitter for radiotherapeutic applications.

Conclusions
We systematically replaced the amino acids (Gln 7 , Trp 8 , Ala 9 , Val 10 , Gly 11 and His 12 ) at potential cleavage sites of the previously reported sequence of [ 68 Ga]Ga-TacBOMB2, and identified that Tle 10 and NMe-His 12 substitutions, either alone or in combination, led to derivatives with comparable/enhanced GRPR binding affinities.In vivo stability and ex vivo biodistribution studies confirmed the improved stability resulted from unnatural amino acid substitutions, which further led to enhanced tumor uptake.With both Tle 10 and NMe-His 12 substitutions, the top candidate [ 68 Ga]Ga-LW01110 has higher in vivo stability, tumor uptake and tumor-to-background uptake ratios than clinically validated [ 68 Ga]Ga-RM2 and [ 68 Ga]Ga-AMBA, and is promising for use for detecting GRPR-expressing tumors with PET.Due to the observed lower pancreas uptake and foreseeable longer tumor retention as being agonists, our optimized sequence, [Tle 10 ,NMe-His 12 ,Thz 14 ]Bombesin(7-14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents.

Fig. 1
Fig. 1 Chemical structures and GRPR binding affinities (K i , mean ± SD, n = 3) of A LW01085 and its derivatives with an unnatural amino acid substitution at B His 12 , C Val 10 , D Ala 9 , E Gln 7 , F Val 10 -Gly 11 , and G Trp 8 .The potential cleavage sites of LW01085 are pointed by black arrows

Fig. 2
Fig. 2 Chemical structures and GRPR binding affinities (K i , mean ± SD, n = 3) of A Ga-TacBOMB2 and its derivatives with an unnatural amino acid substitution at B His 12 , C Val 10 , D Trp 8 , E Val 10 and His 12 , F Gln 7 , Val 10 and His 12 , G Gln 7 , Val 10 , Gly 11 and His 12 , and H Trp 8 , Val 10 and His 12

Table 1
LogD 7.4 values and in vivo stability of GRPR-targeted tracers and are included here for comparison